Our last pre-print of 2024! We created a simple-to-use, temporally-controlled CRISPR/Cas9 toolset that doesn’t compromise between background and induced editing.

We’ve been excited about sharing these fantastic new systems with the community (some of you had a preview at the last CSHL Genome Eng. conf.), and we would love to hear any feedback or answer any questions you might have for us.

The product of a wonderful collaboration across @genentech led by the brilliant duo of Rajini Srinivasan and Tao Sun, with essential guidance from @andrewng_synbio and contributions from many others.

We hope you find this approach as useful as we did, and don’t hesitate to reach out if you have questions or comment!.

And, that worked out every bit as hoped! Shown here is one example of a known phenotype (KDM1a/LSD1 depletion = Vim and/or HLA-I upregulation). More examples in the main text and supplement.

With these bits in place, Valentina executed a focused screen, looking at the loss-of-function phenotypes for ~50 highly-expressed epigenetic modifier genes in a cancer cell line, based on a hypothesis that targeting these would lead to distinct transcriptional changes.

Valentina adapted elements of the CROP-seq vector concept to work some magic with our guide RNA cassette, optimizing for single cell Cas12a RNA library expression/prep, whether delivering 1, 2, or even 4 guide RNAs in tandem.

Expressing FKBP-enAsCas12a w/a guide library (to KO targets) then depleting the enzyme for ~48 hrs via dTag, guide RNA transcripts/barcodes accumulate. Guides are then linked to a phenotype via scRNA-seq.

Long-story short, Cas12a chews up the transcripts used to assign guides (crRNAs) to individual cells. Taking cues from several studies (via @BKleinstiver, @JohnDoench, @jaybradner, Grey, Bock, and more), we adapted enAsCas12a with a dTag-sensitive degron module (FKBP).

First, this was a wonderfully-collaborative effort by several colleagues @genentech . Spearheaded by the ever-brilliant @Pribaikalochka with special callouts to Carolina Galan and Soren Warming.

Have you been interested combining Perturb-Seq screening with Cas12a’s unique capabilities for multiplexed guide RNA expression? Us too! So, we figured out some useful tricks to make it possible, which you can find in our new pre-print. Short thread below:

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